Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene

Abstract

Background: Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes.

Results: Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells.

Conclusion: The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.

Figure 1

The original TIF-IA gene and its two duplicates in the human genome. Exons and introns are marked with numbered boxes and horizontal lines, respectively. Splicing events between exon 1 and 2 are marked with dashed lines. Each of the TIF-IA genes is named according to its relative location on chromosome 16: that is, loci 15, 28, and 21. The relative levels of expression from each locus, as measured by DS Gene^®, are given as percent of total TIF-IA mRNA in P69 cells and shown on the left. Locus 15 is the original full-length gene, containing 18 exons, coding for a 75 kDa protein (651 aa) (see Additional file 3). The transcription start site is marked by an arrow above exon 1 and the stop codon by a stop sign above exon 18. The 3'UTR accounts for ~47% of the mRNA molecule. The Alu element is located in intron 1. Locus 28 is a duplication of the original TIF-IA gene containing the promoter region of the original gene (marked by black box). A deletion of 768 nucleotides in the promoter region reduced its transcription activity. Also, this locus contains two additional deletions with respect to the original gene: a deletion of 3517 nucleotides between intron 10 and intron 12 (between LTR16A1 and Alu-Sg transposed elements) and a deletion of the last exon (exon 18). The last two exons of locus 28 probably originated following a duplication event of exons 3 and 4 of BANP gene (96.6% sequence similarity). The complete BANP gene is also located on chromosome 16. The mRNA synthesized from that site has the potential to encode a protein of 514 aa according to a translation-prediction tool, using the same start codon as that of locus 15. A protein corresponding to the predicted molecular weight was not detected by western blotting analysis (see Additional file 3). Locus 21 is presumably a duplication of locus 28, containing the exact major deletion as that of locus 28 and, in addition, a deletion downstream of exon 15. The last two exons originated from exonization events of LINE-LTR and Alu-Sq transposed elements. Alternative poly-adenylation signal in the last two exons is shown. Locus 21 has the potential to encode a 106 aa protein from the third AUG downstream from the transcription start site. The first AUG potentially encodes a 39-aa polypeptide. Analysis of ESTs and RT-PCR revealed that two RNA molecules are generated from locus 21, as shown in the lower panel (data not shown).

Figure 2

Retrotransposition and exonization of an Alu element in the first intron of TIF-IA gene in locus 21. (A) All mammalians genomes (except for opossum) contain a LINE element (L2) in intron 1 of TIF-IA gene. During primate evolution, an Alu element was inserted into L2. The L2 and Alu elements accumulated mutations leading to exonization (L2-AEx), in which a 3'ss and two alternative 5'ss are recognized by the splicing machinery. Three alternatively spliced isoforms are generated following this exonization: (i) a skipping isoform with no L2-AEx; (ii) selection of a distal 5'ss (termed 5'ss-a), which generates a 180-nt L2-AEx; and (iii) selection of a proximal 5'ss (termed 5'ss-b), leading to exonization of a 370-nt L2-AEx (left to right, respectively). (B) Multiple alignment of TIF-IA splice sites and flanking regions in all three loci. Splice sites are marked with arrows on the top. Exonic and intronic sequences are in uppercase and lower case, respectively. Mutations relative to the original gene (locus 15) are highlighted in pink. 5'ss-a is the distal splice-site and 5'ss-b is the proximal one.

Figure 3

Scanning of diverse normal human tissues, various human cell lines, and chimpanzee blood for Alu-exonization in the TIF-IA genes. (A) Low level of L2-AEx inclusion in normal tissues. cDNA from normal human tissues and cell lines was amplified by PCR analysis with primers directed to exons 1 and 2 of all three loci of TIF-IA genes. No evidence for Alu exonization was observed in chimpanzee blood. Splicing products were separated on 1.5% agarose gel. The three mRNA isoforms are shown on the right; selected PCR products were eluted and sequenced. 293T are epithelial kidney cells. BJ-1 is normal human fibroblast cell line. HeLa are epithelial cervix cells with adenocarcinoma. HT 1080 originated from a fibrosarcoma. MCF-7 is a breast-cancer cell line. P69 was derived by immortalization of human primary prostate epithelial cells. SK MEL-28 is a melanoma cell line and U2OS is an osteosarcoma cell line. (B) Splicing patterns of L2-AEx in various human leukemia cell lines. Total cytoplasmic RNA was extracted from the indicated cell lines. Splicing products were separated on 1.5% agarose gel after RT-PCR analysis, using primers to exons 1 and 2 of all three loci of TIF-IA, to locus 15 alone, to locus 28 alone, and to locus 21 alone. The three mRNA isoforms are shown on the right. Selected splicing products were eluted from the gel and sequenced. Jurkat, Molt-3, and HSB are human T-cell leukemia cell lines. HL-60 is a myeloid cell line. Dami is a megakaryocytic AMKL (acute megakaryoblastic leukemia) non-Down syndrome (DS) cell line. U937 is derived from a human histiocyic lymphoma. MT-4 is a human T-cell lymphoblast line. Dg-75, Raji, and Daudi are Burkitt's lymphoma cell lines. KM H2 is a human Hodgkin's lymphoma cell line. CMK is a megakaryocytic DS. K562 is a chronic myeloid leukemia (CMK) cell line. MEG-01 is a megakaryoblastic cell line. 697 is pre-B ALL cell line. Nalm-6 and REH are human precursor leukemia cell lines.

Figure 4

A single point mutation activates the L2-AEx in wild-type TIF-IA. (A) A schematic description of the TIF-IA mini-gene cloned into pEGFP-C3 vector, which contains the human genomic sequence from exon 1 to 2 of locus 15. The sequences of the splice sites are shown below. (B) The WT mini-gene and the indicated mutants were transfected into 293T cells. Cytoplasmic RNA was extracted and splicing products were separated on 1.5% agarose gel after RT-PCR analysis, using primers specific for the mini-gene RNA. The mRNA isoforms are shown on the right; the difference between the two upper products is due to alternative 5' splicing of the L2-AEx. (C) Similar analysis to panel B. Position -3 of the 3'ss was mutated from G to each of the other three nucleotides. (D) Different selection of the L2-AEx among cells. Transfection of TIF-IA mini-gene with a G → C mutation at position -3 of the 3'ss of the L2-AEx to seven different cell lines (the name of each cell line is indicated above the lane). The four splicing products are illustrated on the right. From bottom to top are: L2-AEx skipped isoform, selection of 5'ss-b Alu-containing exon, intron retention isoform, and unspliced mRNA. U2OS is a human-bone-osteosarcoma epithelial cell line. Du145 is a prostate-cancer cell line. HT1080 is a fibrosarcoma cell line. HepG2 is a hepatoma cell line. HeLa cells are human epithelial cells from a fatal cervical carcinoma. PC3 is a prostate cancer cell line and 293T is a human-embryonic-kidney cell line.

Figure 5

TheGON4Lb gene and its duplicate YY1AP1. (A) Schematic representation of the genomic organization of GON4Lb and YY1AP1. Start codons and stop codons are marked with black arrows and stop signs, respectively. Insertion of an Alu-exon to the transcript of YY1AP1 insert a premature termination codon (PTC) (B) Multiple alignment of the alternative Alu-exon of YY1AP1 and the relative intronic region of GON4L. Splice sites are marked with black arrows. Substitutions and deletions relative to the original gene are marked in pink. (C) Exonization of an Alu exon in YY1AP1, and not in the original gene, as shown by RT-PCR analysis of U2OS cell line. Primers were designed to the flanking exons of each copy.